Background

As an inhibitor of GSDMD, Disulfiram (DSL) can significantly inhibit cell pyroptosis. Cell pyroptosis plays an important role in renal fibrosis.

Methods

HK-2 cells were induced by Lps and ATP to form a pyroptosis model, and the cells were treated by DSL. CCK-8 detected the cell activity. Immunofluorescence (IF) detected the GSDMD. ELISA detected the expression of inflammatory cytokines. Flow cytometry and Western blot detected cell apoptosis and pyroptosis. Collagen type I kit detected collagen secretion, and western blot detected fibrosis marker protein expression. Then, a rat model of unilateral ureteral obstruction (UUO) was established. HE staining detected the degree of renal tissue injury, and Masson staining detected the degree of fibrosis. What’s more, the apoptosis level of tissue cells was detected by TUNEL. And the inflammatory factors in peripheral blood and renal tissue were detected by ELISA. Furthermore, the expression of GSDMD was detected by immunohistochemistry (IHC), and Western blot was used to detect the expression levels of apoptosis and pyroptosis-related proteins in tissues.

Results

It was found that DSL can inhibit the cell membrane perforation of GSDMD-N by inhibiting the cleavage of GSDMD, hence, it inhibited the occurrence of inflammation, cell pyroptosis, and the fibrosis of HK-2 cells. But if the cell has already undergone pyroptosis, DSL does not provide significant prevention. In vivo experiment, it further verified that pretreated DSL had inhibited renal fibrosis injury.

Conclusion

Disulfiram can inhibit inflammation and fibrosis in renal fibrosis rats by inhibiting GSDMD.

中文翻译：

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双硫仑通过抑制 Gasdermin D 裂解和焦亡来抑制大鼠单侧输尿管梗阻模型中的炎症和纤维化

 背景

作为GSDMD的抑制剂，双硫仑（DSL）可以显着抑制细胞焦亡。细胞焦亡在肾纤维化中发挥重要作用。

 方法

Lps和ATP诱导HK-2细胞形成细胞焦亡模型，并用DSL处理细胞。 CCK-8检测细胞活性。免疫荧光 (IF) 检测到 GSDMD。 ELISA检测炎症细胞因子的表达。流式细胞术和Western blot检测细胞凋亡和细胞焦亡。 I型胶原蛋白试剂盒检测胶原蛋白分泌，蛋白质印迹检测纤维化标志蛋白表达。建立大鼠单侧输尿管梗阻(UUO)模型。 HE染色检测肾组织损伤程度，Masson染色检测纤维化程度。并通过TUNEL检测组织细胞的凋亡水平。并采用ELISA法检测外周血和肾组织中的炎症因子。免疫组织化学（IHC）检测GSDMD的表达量，Western blot检测组织中凋亡及细胞焦亡相关蛋白的表达水平。

 结果

研究发现DSL可以通过抑制GSDMD的裂解来抑制GSDMD-N的细胞膜穿孔，从而抑制HK-2细胞炎症、细胞焦亡和纤维化的发生。但如果细胞已经发生焦亡，DSL 并不能提供显着的预防作用。体内实验进一步验证了预处理的DSL具有抑制肾纤维化损伤的作用。

 结论

双硫仑可以通过抑制GSDMD来抑制肾纤维化大鼠的炎症和纤维化。